Gene knockout targeting vector and application thereof
A targeting vector and gene knockout technology, which can be used in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve problems such as easy loss of positive cells, and achieve the effects of convenient cryopreservation and identification, cost reduction, and high efficiency.
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Embodiment 1
[0028] Example 1 Construction of PRNP gene promoter-less targeting vector
[0029] For the construction process of the PRNP gene promoter-free targeting vector, please refer to figure 1 .
[0030] In the present embodiment, get bovine fetal skin tissue and cut into 1mm 3 The left and right small pieces were washed twice with DMEM / F12 and planted in batches in a 25cm medium containing 1mL DMEM / F12+10%FBS. 2 In the culture flask, add DMEM / F12+10% FBS to 6mL after the tissue block is firmly adhered to the wall, and store at 37°C, 5% CO 2 Cultivate in an incubator for 6-7 days, change the medium every 2 days, after the cells grow confluent, digest and passage 2-3 times with 0.25% trypsin, freeze in batches with DMEM / F12+20%FBS+10%DMSO , to obtain bovine fetal fibroblasts.
[0031] Using the genome of bovine fetal fibroblasts as a template, the 5' homology arm and the 3' homology arm were amplified, respectively.
[0032] The primer for the 5' homology arm is, LoxP-5F: 5'-TT ...
Embodiment 2
[0044] Example 2 Transfection of Bovine Fetal Fibroblasts and Cell Screening
[0045] The present invention will knock out the two alleles of the bovine PRNP gene, the liposome cell transfection method is used for the first allele knockout, and the second allele knockout is used Lonza's nuclear electrofection method.
[0046] 2.1 Knockout of the first allele of bovine PRNP
[0047] Inoculate bovine fetal fibroblasts in 6-well cell culture plates and culture them in DMEM medium containing 10% fetal bovine serum to 70-80% confluence. The linearized PRNP gene targeting vector LoxPneo-PRNP KO was transferred into cells;
[0048] After 48 hours of cell transfection, trypsinize the cells from the 6-well plate, inoculate 4×105 cells per 100 mm cell culture dish, and select the cells with G418 concentration of 600 μg / mL cell culture medium for 7-9 days;
[0049] Use a cell cloning ring to select the G418-resistant cell cloning points and inoculate them into a 24-well cell culture p...
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